Near full-genome assay of hcv drug resistance

ABSTRACT

Assays for characterization of genotypic mutations of Hepatitis C Virus (HCV) showing a resistance to anti-HCV drugs.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to assays that detect and characterizesingle or linked mutations in a genome of a Hepatitis C Virus (HCV) thatare associated with resistance of a subject to an anti-HCV drug. Theassays can also be used for predicting resistance to an anti-HCV drug ofa subject infected with HCV prior to or early during antiviral therapyor for selecting an alternative therapy for an HCV-infected subject thathas developed resistance to a particular therapeutic drug or drugcombination. The invention also relates to nucleotide primer pairs andkits for carrying out these assays.

2. Description of Related Art

HCV was cloned and characterized about 15 years ago by Choo andcolleagues. Choo et al. (1989) Science 244, 359-362. HCV belongs to thefamily of Flaviviridae and comprises an enveloped nucleocapsid and asingle-stranded RNA genome of positive polarity. (Bartenschlager et al.(2003) Antiviral Res 60, 91-102.) The HCV genome consists of 5′ and 3′noncoding (UTR or NCR) regions that flank a single long open readingframe (ORF). This ORF encodes three structural proteins at theamino-terminal end and six nonstructural (NS) proteins at thecarboxy-terminal end. The structural proteins are the nucleocapsid coreprotein (C) and the two glycoproteins envelope 1 (E1) and envelope 2(E2). The non-structural proteins are named NS2, NS3, NS4a, NS4b, NS5a,NS5b. The 5′NCR is the most highly conserved region of the HCV genome,whereas the sequences of the two envelope proteins (E1 and E2) arehighly variable among different HCV isolates. The highest degree ofvariation has been observed in a region within E2, now commonly termedhypervariable region 1.

Since the initial identification of HCV, at least 7 different majorviral types have been identified and designated genotype 1 through 7.Within these genotypes are numerous subtypes (e.g. HCV1a, 1b, 1c).Genotype and subtype of a virus with which a subject is infected mayaffect clinical prognosis as well as responsiveness to various drugtreatments. (Simmonds et al. (1995) Hepatology 21, 570-582; Bukh et al.(1995) Semin Liver Dis 15, 41-63; Chevaliez and Pawlotsky (2007) World JGastroenterol 13, 2461-2466).

HCV infection remains a serious medical problem to this date. There arecurrently about 170 million people infected with HCV. HCV is transmittedprimarily by blood and blood products as well as by verticaltransmission during pregnancy. The initial course of infection istypically mild. However, the immune system is often incapable ofclearing the virus, and subjects with persistent infection are at a highrisk for liver cirrhosis and hepatocellular carcinoma. (Poynard et al.(1997) Lancet 349, 825-832).

Current standard treatment for chronic HCV infection is based on acombination of pegylated interferon alpha and ribavirin. This therapyproduces a sustained anti-viral response in 85-90% of subjects infectedwith genotypes 2 and 3, but, unfortunately, only in about 45% ofsubjects infected with the prevalent genotype 1. (Stribling et al.(2006) Gastroenterol Clin North Am vol, 463-486.) Additional therapiesusing other drugs and drug combinations that are endowed with higherantiviral activity and superior safety profiles are clearly required, inparticular for the prevention of HCV recurrence.

Introduction of diagnostic tests for screening blood products hassignificantly reduced the rate of new infection. Availability of invitro models, i.e., HCV subgenomic replicon models and an infectiouscell culture model, and improvements in molecular research techniquessuch as the Polymerase Chain Reaction (PCR) have facilitated developmentof additional potent inhibitors of HCV replication targeting directly aviral protein or acting indirectly through host proteins involved inviral infection. (Bartenschlager (2002) Nat Rev Drug Discov 1, 911-916 ;Wakita et al. (2005) Nat Med 11, 791-796.) Several of these newcompounds have entered clinical trials or are already on the market(http://www.hcvadvocate.org/hepatitis/hepC/HCVDrugs_(—)2007.pdf).

Assays have been developed that are aimed at providing prognosticinformation about the likelihood of responsiveness to an anti-HCVtherapy.(Gretch et al. (1997) Hepatology 26, 43s-47s; Podzorski (2002)Arch Pathol Lab Med 126, 285-290). These assays include serologicaltests and qualitative or quantitative molecular tests. Examples ofPCR-based assays of HCV viral load are Cobas Amplicor® (Roche) and m2000Real-Time PCR Diagnostics System® (Abott). Other PCR-based assays thatinclude, e.g., Versant® HCV Genotyping Assay (Bayer Diagnostics),INNO-LiPA HCV II® (Innogenetics), GEN-ETI-K DEIA kit (Sorin, Saluggia,Italy) and TRUGENE HCV 5′NC genotyping kit (Visible Genetics Europe,Evry, France) identify HCV genotype and subtype. Systematic assessmentof HCV genotype prior to therapy has been advocated recently because HCVgenotype will determine choice and dose regimen of the most effectiveanti-HCV drug, e.g. ribavirin or interferon, as well as duration oftreatment. Current genotype identification relies primarily onsequencing of a small subregion of an HCV genome, e.g., the 5′UTR, butnot of a full or nearly full HCV genome.

SUMMARY OF THE INVENTION

In its most general embodiment, the present invention relates to anassay for identifying a mutation in the genome of an HCV present in asample. The assay comprises the following steps that are carried out insequence:

-   -   a) extraction of viral RNA from the sample containing the HCV;    -   b) determination of genotype and subtype of the HCV;    -   c) synthesis of partial cDNAs of the genome of the HCV in three        separate reverse transcription reactions, the first reverse        transcription reaction initiated from a first outer antisense        primer selected to specifically hybridize to a sequence in the        3′UTR of a prototype HCV genome of the same genotype and        subtype, the second reverse transcription reaction initiated        from a second outer antisense primer selected to specifically        hybridize to a sequence in the NS4B-NS5A region of the genome of        the prototype HCV and the third reverse transcription reaction        initiated from a third outer antisense primer selected to        specifically hybridize to a sequence in the NS2 region of the        genome of the prototype HCV;    -   d) second strand synthesis and amplification of the partial        cDNAs of step c) in three separate PCR reactions, the first PCR        reaction comprising an aliquot of the first reverse        transcription reaction, the first outer antisense primer and a        first outer sense primer selected to specifically hybridize to a        complementary sequence in the NS4B-NS5A region of the genome of        the prototype HCV, the second PCR reaction comprising an aliquot        of the second reverse transcription reaction, the second outer        antisense primer and a second outer sense primer selected to        specifically hybridize to a complementary sequence in the NS2        region of the genome of the prototype HCV, and the third PCR        reaction comprising an aliquot of the third reverse        transcription reaction, the third outer antisense primer and a        third outer sense primer selected to specifically hybridize to a        complementary sequence in the 5′UTR region of the genome of the        prototype HCV, wherein the second outer antisense primer        hybridizes to a sequence in the NS4B-NS5A region of the genome        of the prototype HCV that is located 3′ to the region that is        complementary to the first outer sense primer and wherein the        third outer antisense primer hybridizes to a sequence in the NS2        region of the genome of the prototype HCV that is located 3′ to        the region that is complementary to the second outer sense        primer;    -   e) further amplification of the partial cDNAs of step d) in        three separate nested PCR reactions, the first nested PCR        reaction comprising an aliquot of the first PCR reaction and        first inner antisense and sense primers, the second nested PCR        reaction comprising an aliquot of the second PCR reaction and        second inner antisense and sense primers, and the third nested        PGR reaction comprising an aliquot of the third PCR reaction and        third inner antisense and sense primers, wherein the inner        primers do not overlap the outer primers, the second inner        antisense primer hybridizes to a sequence in the NS4B-NS5A        region of the genome of the prototype HCV that is located 3′ to        the region that is complementary to the first inner sense primer        and the third inner antisense primer hybridizes to a sequence in        the NS2 region of the genome of the prototype HCV that is        located 3′ to the region that is complementary to the second        inner sense primer;    -   f) sequence analysis of the further amplified cDNAs of step e);        and    -   g) comparison of the sequences obtained from step f) with that        of the prototype HCV.

In an another embodiment, an assay of the invention is used to identifyand characterize individual and linked mutations associated withresistance of an HCV-infected subject to particular anti-HCV drugs ordrug combinations and to generate or expand a data bank of HCV mutationsassociated with anti-HCV drug resistance. The assay entails thefollowing steps that are carried out in sequence:

-   -   a) extraction of viral RNA from a sample taken from a subject        carrying an HCV that is resistant to an anti-HCV drug or drug        combination with which the subject has been treated as indicated        by treatment failure;    -   b) determination of genotype and subtype of the HCV;    -   c) synthesis of partial cDNAs of the genome of the HCV in three        separate reverse transcription reactions, the first reverse        transcription reaction initiated from a first outer antisense        primer selected to specifically hybridize to a sequence in the        3′UTR of a prototype HCV genome of the same genotype and        subtype, the second reverse transcription reaction initiated        from a second outer antisense primer selected to specifically        hybridize to a sequence in the NS4B-NS5A region of the genome of        the prototype HCV and the third reverse transcription reaction        initiated from a third outer antisense primer selected to        specifically hybridize to a sequence in the NS2 region of the        genome of the prototype HCV;    -   d) second strand synthesis and amplification of the partial        cDNAs of step c) in three separate PCR reactions, the first PCR        reaction comprising an aliquot of the first reverse        transcription reaction, the first outer antisense primer and a        first outer sense primer selected to specifically hybridize to a        complementary sequence in the NS4B-NS5A region of the genome of        the prototype HCV, the second PCR reaction comprising an aliquot        of the second reverse transcription reaction, the second outer        antisense primer and a second outer sense primer selected to        specifically hybridize to a complementary sequence in the NS2        region of the genome of the prototype HCV, and the third PCR        reaction comprising an aliquot of the third reverse        transcription reaction, the third outer antisense primer and a        third outer sense primer selected to specifically hybridize to a        complementary sequence in the 5′UTR region of the genome of the        prototype HCV, wherein the second outer antisense primer        hybridizes to a sequence in the NS4B-NS5A region of the genome        of the prototype HCV that is located 3′ to the region that is        complementary to the first outer sense primer and wherein the        third outer antisense primer hybridizes to a sequence in the NS2        region of the genome of the prototype HCV that is located 3′ to        the region that is complementary to the second outer sense        primer;    -   e) further amplification of the partial cDNAs of step d) in        three separate nested PCR reactions, the first nested PCR        reaction comprising an aliquot of the first PCR reaction and        first inner antisense and sense primers, the second nested PCR        reaction comprising an aliquot of the second PCR reaction and        second inner antisense and sense primers, and the third nested        PCR reaction comprising an aliquot of the third PCR reaction and        third inner antisense and sense primers, wherein the inner        primers do not overlap the outer primers, the second inner        antisense primer hybridizes to a sequence in the NS4B-NS5A        region of the genome of the prototype HCV that is located 3′ to        the region that is complementary to the first inner sense primer        and the third inner antisense primer hybridizes to a sequence in        the NS2 region of the genome of the prototype HCV that is        located 3′ to the region that is complementary to the second        inner sense primer;    -   f) sequence analysis of the further amplified cDNAs of step e);    -   g) comparison of the sequences obtained from step f) with that        of the prototype HCV and identification of mutations; and    -   h) entry of the mutations identified in a data bank of HCV        mutations associated with anti-HCV drug resistance.

In a related embodiment, an assay of the invention is employed toidentify and characterize individual and linked mutations associatedwith resistance of an HCV-infected subject to the treatment administeredand, making use of a data bank of HCV mutations associated with anti-HCVdrug resistance to select an alternative anti-HCV drug or drugcombination to which the virus variant of the subject is not expected tobe resistant for further therapy of the subject. The assay comprises thesame steps as that of the preceding embodiment, except for step h) thatis replaced by a step entailing a search of a data bank of HCV mutationsassociated with anti-HCV drug resistance for the mutations identifiedand selection for subsequent treatment of the subject of an anti-HCVdrug or drug combination to which the HCV is not expected to beresistant.

In another embodiment, the assay of the preceding embodiment is employedfor analysing a sample taken from a subject infected with HCV prior tocommencement of any pharmacological therapy of the subject. Informationobtained from this analysis will permit a treating physician to selectan anti-HCV drug or drug combination for treatment of the subject, towhich anti-HCV drug or drug combination the HCV variant or variantspresent in the subject are not expected to be resistant.

A more specific embodiment of the invention relates to an assay foridentifying a mutation in the genome of an HCV1b present in a sample.The assay comprises the following steps that are carried out insequence:

-   -   a) extraction of viral RNA from the sample containing the HCV;    -   b) determination of genotype and subtype of the HCV;    -   c) provided that step b) indicated that the HCV is of type 1b,        synthesis of partial cDNAs of the genome of the HCV in three        separate reverse transcription reactions, the first reverse        transcription reaction initiated from primer poly-A, the second        reverse transcription reaction initiated from primer HCV1bOR6312        and the third reverse transcription reaction initiated from        primer HCV1bOR3306;    -   d) second strand synthesis and amplification of the partial        cDNAs of step c) in three separate PCR reactions, the first PCR        reaction comprising an aliquot of the first reverse        transcription reaction and primer pair poly-A/HCV1bOF6074, the        second PCR reaction comprising an aliquot of the second reverse        transcription reaction and primer pair HCV1bOR6312/HCV1bOF1977        and the third PCR reaction comprising an aliquot of the third        reverse transcription reaction and primer pair        HCV1bOR3306/HCVOF129;    -   e) further amplification of the partial cDNAs of step d) in        three separate nested PCR reactions, the first nested PCR        reaction comprising an aliquot of the first PCR reaction and        primer pair HCV1bIR9339/HCV1bIF6126, the second nested PCR        reaction comprising an aliquot of the second PCR reaction and        primer pair HCV1bIR6282/HCV1bIF2523, and the third nested PCR        reaction comprising an aliquot of the third PCR reaction and        primer pair HCV1bIR2770/HCVIF278;    -   f) sequence analysis of the further amplified cDNAs of step e);        and    -   g) comparison of the sequences obtained from step f) with that        of a prototype HCV1b.

In another embodiment, an assay of the invention is used to identify andcharacterize individual and linked mutations associated with resistanceof a subject infected with an HCV1b to a particular anti-HCV drug ordrug combination and to generate or expand a data bank of HCV mutationsassociated with anti-HCV drug resistance. The assay entails thefollowing steps that are carried out in sequence:

-   -   a) extraction of viral RNA from a sample taken from a subject        harboring an HCV that is resistant to an anti-HCV drug or drug        combination with which the subject has been treated;    -   b) determination of genotype and subtype of the HCV;    -   c) provided that step b) indicated that the HCV is of type 1b,        synthesis of partial cDNAs of the genome of the HCV in three        separate reverse transcription reactions, the first reverse        transcription reaction initiated from primer poly-A, the second        reverse transcription reaction initiated from primer HCV1bOR6312        and the third reverse transcription reaction initiated from        primer HCV1bOR3306;    -   d) second strand synthesis and amplification of the partial        cDNAs of step c) in three separate PCR reactions, the first PCR        reaction comprising an aliquot of the first reverse        transcription reaction and primer pair poly-A/HCV1bOF6074, the        second PCR reaction comprising an aliquot of the second reverse        transcription reaction and primer pair HCV1bOR6312/HCV1bOF1977        and the third PCR reaction comprising an aliquot of the third        reverse transcription reaction and primer pair        HCV1bOR3306/HCVOF129;    -   e) further amplification of the partial cDNAs of step d) in        three separate nested PCR reactions, the first nested PCR        reaction comprising an aliquot of the first PCR reaction and        primer pair HCV1bIR9339/HCV1bIF6126, the second nested PCR        reaction comprising an aliquot of the second PCR reaction and        primer pair HCV1bIR6282/HCV1bIF2523, and the third nested PCR        reaction comprising an aliquot of the third PCR reaction and        primer pair HCV1bIR2770/HCVIF278;    -   f) sequence analysis of the further amplified cDNAs of step e);    -   g) comparison of the sequences obtained from step f) with that        of a prototype HCV1b and identification of mutations; and    -   h) entry of the mutations identified in a data bank of HCV        mutations associated with anti-HCV drug resistance.

Once a useful data bank has been assembled, a similar assay can beutilized to analyze samples from treatment-naïve HCV1b-infected subjectsor from subjects infected with HCV1b that have been treated anddeveloped resistance to the treatment regimen to select an appropriateanti-HCV drug or drug combination for treatment or further treatment,respectively. In such embodiments, step h) of the assay describedimmediately above is replaced by a step entailing a search of a databank of HCV mutations associated with anti-HCV drug resistance for themutations identified and selection for subsequent treatment of thesubject of an anti-HCV drug or drug combination to which the HCV is notexpected to be resistant.

The invention also relates to primer pairs consisting of poly-A andHCV1bOF6074, HCV1bOR6312 and HCV1bOF1977, HCV1bOR3306 and HCVOF129,HCV1bIR9339 and HCV1bIF6126, HCV1bIR6282 and HCV1bIF2523, andHCV1bIR2770 and HCVIF278. Kits for detecting mutations in an HCV genomeare also an object of the invention. These kits comprise at least one orall of the aforementioned primer pairs and can include additionalreagents such as e.g., polymerase, buffers, and nucleosidetriphosphates.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically represents the partial cDNA synthesis andamplifcation steps of assays of the invention on the example of an HCVtype 1b.

FIG. 2 compares the sensitivity of the partial cDNA amplification methodof the invention with that of amplifcation of full-length viral genomes.

FIGS. 3 a and b compare translated HCV amino acid sequences from twoinfected subjects obtained by an assay of the invention with thesequence of a prototype HCV genome.

DETAILED DESCRIPTION OF THE INVENTION

To aid in understanding the invention, several terms are defined below.

The terms “nucleic acid” and “oligonucleotide” refer to primers andoligomer fragments to be amplified or detected, and shall be generic topolydeoxyribonucleotides (containing 2-deoxy-D-ribose), also named DNA,to polyribonucleotides (containing D-ribose), also named RNA, and to anyother type of polynucleotide which is an N glycoside of a purine orpyrimidine base, or modified purine or pyrimidine base. There is nointended distinction in length between the terms “nucleic acid” and“oligonucleotide”, and these terms will be used interchangeably. Theseterms refer only to the primary structure of the molecule. Thus, theseterms include double- and single-stranded DNA, as well as double- andsingle-stranded RNA.

The term “cDNA” refers to complementary DNA which is DNA synthesizedfrom an RNA template by the action of RNA-dependent DNA polymerase orreverse transcriptase or DNA polymerase. These terms include double- andsingle-stranded complementary DNA.

Oligonucleotides can be prepared by any suitable method, including, forexample, cloning and restriction of appropriate sequences and directchemical synthesis by a method such as the phosphotriester method ofNarang et al. (1979) Meth Enzymol 68, 90-99; the phosphodiester methodof Brown et al. (1979) Meth Enzymol 68, 109-151; thediethylphosphoramidite method of Beaucage et al. (1981) Tetrahedron Lett22, 1859-1862; and the solid support method of U.S. Pat. No. 4,458,066.

The terms “hybridization” and “hybridize” refer to the formation of aduplex structure by two single-stranded nucleic acids due tocomplementary base pairing. Hybridization can occur between fully(exactly) complementary nucleic acid strands or between “substantiallycomplementary” nucleic acid strands that contain minor regions ofmismatch. Conditions under which only fully complementary nucleic acidstrands will hybridize are referred to as “stringent hybridizationconditions”. Stable duplexes of substantially complementary sequencescan be achieved under less stringent hybridization conditions. Thoseskilled in the art of nucleic acid technology can determine duplexstability empirically following the guidance provided by the art (see,e.g., Sambrook et al. (1985) Molecular Cloning—A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.). Generally,stringent conditions are selected to be about 5o C lower than thethermal melting point (Tm) for the specific sequence at a defined ionicstrength and pH. The Tm is the temperature (under defined ionic strengthand pH) at which 50% of the base pairs have dissociated. Relaxing thestringency of the hybridization conditions will allow sequencemismatches to be tolerated; the degree of mismatch tolerated can becontrolled by suitable adjustment of the hybridization conditions.Hybridization of both exactly complementary and substantiallycomplementary nucleic acid strands is referred to herein as “specific”.

The term “primer” refers to an oligonucleotide capable of acting as apoint of initiation of DNA synthesis under conditions in which synthesisof a primer extension product complementary to a nucleic acid strand isinduced, i.e., in the presence of four different nucleosidetriphosphates and an agent for polymerization (i.e., DNA polymerase orreverse transcriptase) in an appropriate buffer and at a suitabletemperature. A primer is preferably a single-strandedoligodeoxyribonucleotide. The appropriate length of a primer depends onthe intended use of the primer but typically ranges from about 15 toabout 35 nucleotides. Short primer molecules generally require coolertemperatures to form sufficiently stable hybrid complexes with thetemplate. A primer need not reflect the exact sequence of the templatenucleic acid, but must be sufficiently complementary to hybridize withthe template. Primers can incorporate additional features which allowfor the detection or immobilization of the primer but do not alter thebasic property of the primer, that of acting as a point of initiation ofDNA synthesis. The region of the primer which is sufficientlycomplementary to the template to hybridize is referred to herein as thehybridizing region.

As used herein, a “sense” or “upstream” primer refers to a primer whoseextension product is a subsequence of the coding strand; an “antisense”or “downstream” primer refers to a primer whose extension product is asubsequence of the complementary non-coding strand.

The terms “outer primer” or “outer primers” refer to the first primer orpair of primers that are used to reverse-transcribe or initially amplifya stretch of a nucleic acid. The terms “inner primer” or “inner primers”refer to a primer or to a pair of primers that are used to furtheramplify the initial amplification product. Inner primers do not sharesignificant sequence homology with corresponding outer primers, andamplification by inner primers typically produces a secondaryamplification product that is slightly shorter than the initialamplification product.

As used herein, an oligonucleotide primer is “specific” for a targetsequence if the number of mismatches present between the oligonucleotideand the target sequence is less than the number of mismatches presentbetween the oligonucleotide and non-target sequences. Hybridizationconditions can be chosen under which stable duplexes are formed only ifthe number of mismatches present is no more than the number ofmismatches present between the oligonucleotide and the target sequence.Under such conditions, the target-specific oligonucleotide can form astable duplex only with a target sequence. The use of target-specificprimers under suitably stringent amplification conditions enables thespecific amplification of those target sequences that contain the targetprimer binding sites.

The terms “target region” and “target nucleic acid” refers to a regionof a nucleic acid that is to be amplified or otherwise analyzed. Thesequence to which a primer hybridizes can be referred to as a “target”.

The terms “variant” or “HCV variant” refers to an HCV having a genomethat differs from that of the corresponding prototype virus by thepresence of at least one mutation that causes at least a one amino acidchange in a viral protein product. Accordingly, the term “mutation”refers to such changes in the genome of a variant that result in a viralprotein product that differs from that of the prototype virus by atleast one amino acid.

The term “prototype virus” or “prototype HCV” means a reference HCVvirus that provides a reference genome with which the viral sequencesproduced in the assays of the invention are compared and which serve asa basis for designing the cDNA synthesis, amplification and sequencingprimers used in the assays.

Prototype virus may refer to a specific virus isolate. Alternatively, itmay refer to a hypothetical HCV virus that contains a consensus genomederived from comparison of genomic sequences of multiple virus isolates.One such prototype virus, HCV strain H77 (Genbank Accession NumberAF011751), was used in example 1 for designing HCV 1b primers. HCV cont(Genbank Accession Number AJ238799) was used as prototype HCV 1b genomicsequence in the assays of example 3. A person skilled in the art willknow how to select prototype HCV sequences for other genotypes andsubtypes. For example, HCV strains HC-J6 (Genbank Accession NumberD00944) or JFH1 (Genbank Accession Number AB047639) may be considered asprototype HCV 2a.

The term “quasispecies” relates to a group of highly related HCV ofidentical genotype and subtype frequently present in an infectedsubject.

The terms “anti-HCV drug or drug combination” refer to any drug or drugcombination, respectively, that is capable of decreasing HCV viral loador viral titer in at least a subset of HCV-infected subjects. Inparticular, these terms also refer to any drug or drug combination,respectively, that is capable of substantially or significantlydecreasing HCV viral load or viral titer in at least a subset ofHCV-infected subjects. Anti-HCV drugs include but are not limited to HCVreplication inhibitors such as polymerase inhibitors, proteaseinhibitors and cyclophilin inhibitors, immune or host responsemodulators, virus entry inhibitors and host factor inhibitors.

The term “data bank of HCV mutations associated with anti-HCV drugresistance” refers to a compilation, in any form, of mutations that areassociated with resistance to any anti-HCV drug or drug combination,resistance being indicated by a failure of a course of treatment with ananti-HCV drug or drug combination to substantially reduce viral load(also referred to as treatment failure). Such a data bank may beassembled using information available in the art or becoming availablein the art as well as information obtained from analyses of samples fromdrug-resistant, infected subjects conducted employing the assays of thepresent invention.

Presently available assays do not indicate whether a member of apopulation of HCV variants present in a subject (quasispecies) or evenwhether a predominant variant will be resistant to a treatment with aparticular anti-HCV drug or a combination of anti-HCV drugs. Moreover,these tests do not determine which individual mutations, linkedmutations or fingerprint within a viral genome are associated with viralresistance to a particular anti-HCV therapy. Most therapies against HCVare very expensive and lengthy. Treating an HCV-infected subject withoutknowing whether it is a priori resistant to the particular anti-HCV drugor drug combination utilized may result in adverse effects in thesubject due to the consequences of the continued presence of virus athigh levels as well as to secondary effects (toxicity) of the anti-HCVtreatment.

A diagnostic assay for characterizing mutations in a complete or nearlycomplete HCV genome of a particular genotype and/or subtype that areassociated with resistance to a particular anti-HCV drug or drugcombination is a long felt need for the person skilled in the art, i.e.a physician, a clinician or a nurse at a hospital or medical carefacility. The present invention provides such an assay. The assay relieson amplification by reverse transcription—polymerase chain reaction(RT-PCR) of an HCV genome as three overlapping DNA fragments of similarlengths. The inventors found that amplification of the genome as threefragments represents a best compromise between the need for sensitivityof the assay and avoidance of selection of viral variant sequences.Within limits, sensitivity of the assay would increase with the numberof discrete fragments amplified, whereas minimization of selection wouldrequire a decrease in the number of discrete fragments amplified. Theassays of the invention are highly sensitive and, in contrast to assaysbased on reverse transcription and amplification of full-length viralgenomes, permit detection and analysis of HCV genomes from samples takenfrom subjects with very low viral loads.

The assays of the invention will enable the systematic assembly of adata bank of mutations that are associated with resistance to particularanti-HCV drugs and, once a useful data set has been assembled, can beemployed as prognostic assays for determining the presence in aninfected subject of an HCV variant that is resistant to treatment with aparticular anti-HCV drug(s). Based on such information, it will bepossible to select an appropriate anti-HCV drug or drug combination fora therapy of the infected subject that will not be hampered by drugresistance of a detectable HCV variant already present in the subjectprior to therapy. Moreover, once a particular therapeutic regimen isselected and treatment of the subject has been initiated, virus isolatedfrom the subject could be introduced into an infectious cell culturemodel and allowed to undergo a few rounds of replication under theselective pressure of the drug or drug combination used on the subject.An assay of the invention could then be performed on the selected viruspopulation to determine whether amplification had occurred of a minordrug-resistant variant that could not be detected prior to suchamplification. Results obtained could be utilized to rapidly adapt orchange the drug regimen administered to the subject.

In addition to measurements of viral load, an assay of the inventioncould be employed for monitoring development of drug resistance in asubject treated with an anti-HCV drug or drug combination. Virus wouldbe isolated at the end of a course of therapy or at various times duringtreatment and analysed using an assay of the invention. Detection of amajor variant containing a known resistance mutation or set of linkedmutations would provide an indication, which is independent fromdeterminations of viral load, that the therapeutic regimen needs beadapted or replaced by another regimen. The above-mentioned data bank ofmutations would assist the treating physician in the choice of anadapted or alternative regimen.

The assays of the present invention are highly sensitive and enabledetection, by a single procedure, of individual mutations or linkedmutations occurring essentially anywhere in an HCV genome that areassociated with resistance to a treatment with any combination of one ormore anti-HCV drugs.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, polypeptide and nucleic acid synthesis, and immunology,which are within the skill of the art. Such techniques are explainedfully in the literature. See e.g., Sambrook et al., (1989) MolecularCloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.; DNA Cloning—a Practical Approach, volumes 1 and 2 (D. M.Glover, ed., 1985); Oligonucleotide Synthesis (M. J. Gait, ed., 1984);Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins, eds., 1984);Transcription and Translation (B. D. Hames & S. J. Higgins, eds., 1984);Animal Cell Culture—a Practical Approach (R. I. Freshney, ed., 1986);Immobilized Cells and Enzymes—A Practical Approach (J. Woodward, ed.,1985); B. Perbal (1984) A Practical Guide to Molecular Cloning, JohnWiley & Sons, New York, N.Y.; the series Methods in Enzymology, AcademicPress, Inc., including volume 154 (R. Wu & L. Grossman, eds., 1987) andvolume 155 (R. Wu, ed., 1987); Gene Transfer Vectors for Mammalian Cells(J. H. Miller & M. P. Calos, eds., 1987); Immunochemical Methods in Celland Molecular Biology (R. J. Mayer & J. H. Walker, eds., 1987), ProteinPurification—Principles and Practice (R. K. Scopes, 1987); and Handbookof Experimental immunology, volumes 1-4 (D. M. Weir & C. C. Blackwell,eds., 1986).

In the most general embodiment, the present invention relates to anassay that is capable of identifying and characterizing single andlinked mutations in a genome of an HCV variant present in a sample, theassay comprising the following steps that are carried out sequentially:

-   -   Step 1: extraction of viral RNA from a sample containing an HCV;    -   Step 2: determination of genotype and subtype of the HCV;    -   Step 3: synthesis of partial cDNAs of the genome of the HCV in        three separate reverse transcription reactions, the first        reverse transcription reaction initiated from a first outer        antisense primer selected to specifically hybridize to a        sequence in the 3′UTR of a prototype HCV of the same genotype        and subtype, the second reverse transcription reaction initiated        from a second outer antisense primer selected to specifically        hybridize to a sequence in the NS4B-NS5A region of the genome of        the prototype HCV and the third reverse transcription reaction        initiated from a third outer antisense primer selected to        specifically hybridize to a sequence in the NS2 region of the        genome of the prototype HCV;    -   Step 4: second strand synthesis and amplification of the partial        cDNAs of the genome of the HCV in three separate PCR reactions,        the first PCR reaction comprising an aliquot of the first        reverse transcription reaction, the first outer antisense primer        and a first outer sense primer selected to specifically        hybridize to a complement of a sequence in the NS4B-NS5A region        of the genome of the prototype HCV, the second PCR reaction        comprising an aliquot of the second reverse transcription        reaction, the second outer antisense primer and a second outer        sense primer selected to specifically hybridize to a complement        of a sequence in the NS2 region of the genome of the prototype        HCV, and the third PCR reaction comprising an aliquot of the        third reverse transcription reaction, the third outer antisense        primer and a third outer sense primer selected to specifically        hybridize to a complement of a sequence in the 5′UTR region of        the genome of the prototype HCV, wherein the second outer        antisense primer hybridizes to a sequence in the NS4B-NS5A        region of the genome of the prototype HCV that is located 3′ to        the region that is complementary to the first outer sense primer        and wherein the third outer antisense primer hybridizes to a        sequence in the NS2 region of the genome of the prototype HCV        that is located 3′ to the region that is complementary to the        second outer sense primer;    -   Step 5: further amplification of the partial cDNAs of step 4 in        three separate nested PCR reactions, the first nested PCR        reaction comprising an aliquot of the first PCR reaction and        first inner antisense and sense primers, the second nested PCR        reaction comprising an aliquot of the second PCR reaction and        second inner antisense and sense primers, and the third nested        PCR reaction comprising an aliquot of the third PCR reaction and        third inner antisense and sense primers, wherein the inner        primers do not overlap the outer primers, the second inner        antisense primer hybridizes to a sequence in the NS4B-NS5A        region of the genome of the prototype HCV that is located 3′ to        the region that is complementary to the first inner sense primer        and the third inner antisense primer hybridizes to a sequence in        the NS2 region of the genome of the prototype HCV that is        located 3′ to the region that is complementary to the second        inner sense primer;    -   Step 6: sequence analysis of the further amplified cDNAs of step        5; and    -   Step 7: comparison of the sequences obtained with that of the        prototype HCV.

In a more specific embodiment, the present invention relates to an assaythat is capable of identifying and characterizing single and linkedmutations in a genome of an HCV variant resistant to an anti-HCV drug ordrug combination in a sample taken from a subject that has beenundergoing treatment with the anti-HCV drug or drug combination. Theassay comprises the following steps that are carried out sequentially:

-   -   Step 1: extraction of viral RNA from a sample taken from a        subject harboring an HCV that is resistant to an anti-HCV drug        or drug combination with which the subject has been treated;

Steps 2-6 as in the assay described above;

-   -   Step 7: comparison of the sequences obtained in step 6 with that        of the genome of the prototype HCV and identification of        mutations; and    -   Step 8: entry of the mutations identified in a data bank of        mutations associated with anti-HCV drug resistance.

Once a useful data bank of mutations associated with drug resistance hasbeen established, the present invention will also relate to an assaythat is identical to the assay described immediately before, except thatstep 8 will consist of a search of the data bank for the mutationsidentified and selection of an alternative anti-HCV drug or drugcombination to which the HCV is not expected to be resistant forsubsequent treatment of the subject.

The latter assay of the invention may also be carried out on a sampletaken from a subject infected with HCV prior to commencement of anypharmacological therapy of the subject. Results from the assay willallow the physician to select an anti-HCV drug or drug combination towhich the HCV the subject is infected with is not resistant. It isrealized that a subject frequently is infected with a quasispecies ofHCV comprising a dominant variant and multiple, minor variants. Theassay primarily will uncover mutations present in the dominant variant.Whether mutations present in minor variants will also be discoverablewill depend on multiple factors, in particular on the relative abundanceof minor variants and the sequencing technology employed. Mutations in aminor variant should be identifiable by routine capillary sequencing,i.e., standard automated sequencing, if the relative abundance of theminor variant is at least about 20%. If representation of a variant islower, an advanced sequencing methodology such as high-throughputsequencing-by-synthesis technologies will need to be utilized to obtainmutational information. Such sequencing technologies were developed,e.g., by Illumina Inc., San Diego, Calif., 454 Life Sciences, Branford,Conn., and Applied Biosciences, Foster City, Calif. Sequencing equipmentand services are commercially available.

Specific embodiments of assays for use with samples containing an HCV 1bvariant are presented below and are further illustrated in the Examplesection. One such assay comprises the following steps that are carriedout in sequence:

-   -   Step 1: extraction of viral RNA from a sample containing an HCV;    -   Step 2: determination of genotype and subtype of the HCV;    -   Step 3: provided that step 2 indicated that the HCV is of type        1b, synthesis of partial cDNAs of the genome of the HCV in three        separate reverse transcription reactions, the first reverse        transcription reaction initiated from primer poly-A, the second        reverse transcription reaction initiated from primer HCV1bOR6312        and the third reverse transcription reaction initiated from        primer HCV1bOR3306;    -   Step 4: second strand synthesis and amplification of the partial        cDNAs of the genome of the HCV in three separate PCR reactions,        the first PCR reaction comprising an aliquot of the first        reverse transcription reaction and primer pair        poly-A/HCV1bOF6074, the second PCR reaction comprising an        aliquot of the second reverse transcription reaction and primer        pair HCV1bOR6312/HCV1bOF1977 and the third PCR reaction        comprising an aliquot of the third reverse transcription        reaction and primer pair HCV1bOR3306/HCVOF129;    -   Step 5: further amplification of the partial cDNAs of step 4 in        three separate nested PCR reactions, the first nested PCR        reaction comprising an aliquot of the first PCR reaction and        primer pair HCV1bIR9339/HCV1bIF6126, the second nested PCR        reaction comprising an aliquot of the second PCR reaction and        primer pair HCV1bIR6282/HCV1bIF2523, and the third nested PCR        reaction comprising an aliquot of the third PCR reaction and        primer pair HCV1bIR2770/HCVIF278;    -   Step 6: sequence analysis of the further amplified cDNAs of step        5; and    -   Step 7: comparison of the sequences obtained with that of a        prototype HCV1b.

Another such assay for use with samples obtained from a subjectharboring an HCV1b that is resistant to the therapy the subject hasreceived comprises the following steps that are carried out in sequence:

-   -   Step 1: extraction of viral RNA from a sample taken from a        subject infected with an HCV that is resistant to an anti-HCV        drug or drug combination with which the subject has been        treated;    -   Steps 2-6 as in the preceding assay;    -   Step 7: comparison of the sequences obtained in step 6 with that        of the genome of the prototype HCV and identification of        mutations; and    -   Step 8: entry of the mutations identified in a data bank of        mutations associated with anti-HCV drug resistance.

A similar assay that can be carried out once a useful data bank of HCV1bmutations associated with drug resistance has been generated isidentical to the assay described immediately before, except that step 8will consist of a search of the latter data bank for the mutationsidentified and selection of an alternative anti-HCV drug or drugcombination to which the HCV is not expected to be resistant forsubsequent treatment of the subject.

The above assay may also be carried may also be carried out on a sampletaken from a subject infected with HCV prior to commencement of anypharmacological therapy of the subject. Provided that the HCV of thesubject is of type 1b, results from the assay would allow the physicianto select an anti-HCV drug or drug combination to which the HCV thesubject is infected with is not resistant.

Key aspects of the above HCV1b-specific assays are also illustrated inFIG. 1 and in the Example section. The nucleotide sequences of cDNAsynthesis and amplification primers as well as a set of proposedsequencing primers are provided in Table 1.

TABLE 1 HCV 1b primers Location in the genome (H77 as SEQ ID Primer namePurpose Direction Primer sequence reference) NO: HCV1bOR3306cDNA/outer PCR antisense GATGATGTCCCCAC 3332-3306 3 5′UTR-NS2 regionACGCCGCGGTGTC HCVOF129 outer PCR 5′UTR- sense CCGGGAGAGCCAT 129-157 6NS2 region AGTGGTCTGCGGA ACC HCVIF278 inner PCR 5'UTR- senseGCCTTGTGGTACTG 278-307 12 NS2 region CCTGATAGGGTGCT TG HCV1bIR2770inner PCR 5'UTR- antisense TCCGCACGATGCA 2798-2770 9 NS2 regionGCCATCTCCCGGTC CA HCV1bOR6312 cDNA/outer PCR antisense GGCAGGAGCTTGG6343-6312 2 NS2-NS5A region ACTGGAGCCAGGT CTTGAA HCV1bOF1977outer PCR NS2- sense CAAGGCAACTGGTT 1977-2008 5 NS5A regionCGGCTGTACATGGA TGAA HCV1bIF2523 inner PCR NS2- sense GACGCGCGCGTCT2523-2551 11 NS5A region GYGCCTGCTTRTGG AT HCV1bIR6282 inner PCR NS2-antisense TCAGTCAACACCGT 6310-6282 8 NS5A region GCATATCCAGTCCC A poly-AcDNA/outer PCR antisense 30-mer 9447-9418 1 NS4B-3′UTR region HCVOF6074outer PCR NS4B- sense GGCTGTGCAGTGG 6074-6103 4 3′UTR regionATGAACCGGCTGAT AGC HCV1bIF6126 inner PCR NS4B- sense GTCTCCCCCACGCA6126-6151 10 3′UTR region CTATGTGCCTGA HCV1bIR9339 inner PCR NS4B-antisense  GGGAGCAGGTAGA 9367-9342 7 3′UTR region TGCCTACCCCTACHCV1bSF310 sequencing 5′UTR- sense GAGTGCCCCGGGA 309-332 13 NS2 regionGGTCTTCGTAGA HCV1bSF807 sequencing 5′UTR- sense CCGGGTTCTGGAG 806-829 14NS2 region GACGGCGTGAA HCV1bSR850 sequencing 5′UTR- antisenseAGGAAGATAGAGAA 874-849 15 NS2 region AGAGCAACCGGG HCV1bSF1202sequencing 5′UTR- sense TCTCCCAGCTGTTC 1201-1222 16 NS2 region ACCTTCTCHCV1bSR1302 sequencing 5′UTR- antisense GACCAGTTCATCAT 1321-1301 17NS2 region CATATCC HCV1bSF1597 sequencing 5′UTR- sense GGCAGCTGGCACA1593-1615 18 NS2 region TCAACAGGAC HCV1bSR1653 sequencing 5′UTR-antisense GTAGAACAGCGCG 1674-1653 19 NS2 region GCAAGGAAC HCV1bSF1854sequencing 5′UTR- sense TGGTCCAGTGTATT 1850-1872 20 NS2 region GYTTCACCCHCV1bSR1990 sequencing 5′UTR- antisense TTCATCCATGTACA 2008-1986 21NS2 region GCCGAACCA HCV1bSF2242 sequencing 5′UTR- sense GTGGGGGGCGTGG2238-2259 22 NS2 region AGCACAGGC HCV1bSR2433 sequencing 5′UTR-antisense CGTACAGGTATTGC 2451-2429 23 NS2 region ACGTCCACG HCV1bSF2640sequencing NS2- sense TCTCTCCTTCCTTG 2636-2658 24 NS5A region TGTTCTTCTHCV1bSF2860 sequencing NS2- antisense AACCACCATATGAG 2882-2860 25NS5A region CCTGGCGAG HCV1bSF3085 sequencing NS2- sense CGCGCTCAAGGGC3081-3102 26 NS5A region TCATYCGTG HCV1bSR3280 sequencing NS2- antisenseCAGGTGATGATCTT 3298-3276 27 NS5A region GGTCTCCAT HCV1bSF3541sequencing NS2- sense ACRCAATCTTTCCT 3537-3559 28 NS5A region GGCGACCTGHCV1bSR3643 sequencing NS2- antisense GTCCTGGTCTACAT 3662-3639 29NS5A region TGGTGTACAT HCV1bSF4004 sequencing NS2- sense CGCAGACATTCCAA4000-4021 30 NS5A region GTGGCCCA HCV1bSR4237 sequencing NS2- antisenseCAACCACCGTCGG 4255-4233 31 NS5A region CAAGGAACTT HCV1bSF4516sequencing NS2- sense CTCATTTTCTGCCA 4512-4534 32 NS5A region TTCCAAGAAHCVlbSR4666 sequencing NS2- antisense TCAAAGTCGCCGGT 4687-4662 33NS5A region AWAGCCCGTCAT HCV1bSF5048 sequencing NS2- senseTAGATGCCCACTTC 5044-5064 34 NS5A region TTGTCCC HCV1bSR5164sequencing NS2- antisense AGCCGTATGAGACA 5182-5160 35 NS5A regionCTTCCACAT HCV1bSF5524 sequencing NS2- sense CAATTCAAGCAGAA 5520-5542 36NS5A region GGCGCTCGG HCV1bSR5639 sequencing NS2- antisenseTGTATCCCGCTGAT 5659-5635 37 NS5A region GAARTTCCACA HCV1bSF5986sequencing NS2- sense ACACGCTGTGATAA 5986-6008 38 NS5A regionATGTCTCCCCCGC HCV1bSR6092 sequencing NS2- antisense GAAGCGAACGCTAT6112-6088 39 NS5A region CAGCCGGTTCA HCV1bSF6186 sequencing NS2- senseCCTCTCCAGCCTTA 6182-6205 40 NS5A region CCATCACTCA HCV1bSR6256sequencing NS2- antisense TCCCTIAGCCACGA 6281-6256 41 NS5A regionGCCGGAGCATGG HCV1bSF6416 sequencing NS4B- sense TCATGCAIACCACCT6416-6437 42 3′UTR region GCCCATG HCV1bSR6616 sequencing NS4B- antisenseTCCCCCACCCGCG 6639-6616 43 3′UTR region TGACCTCCAC HCV1bSF6853sequencing NS4B- sense GTGCTCACTTCCAT 6849-6871 44 3′UTR regionGCTCACCGA HCV1bSR6964 sequencing NS4B- antisense TCAAGGAAGGCGC 6975-695445 3′UTR region AGACAACTG HCV1bSF7066 sequencing NS4B- senseGGGAACATCACCC 7056-7078 46 3′UTR region GCGTGGAGTC HCV1bSR7273sequencing NS4B- antisense GGGCACCCGTGTA 7285-7263 47 3′UTR regionCCACCGGAGG HCV1bSF7504 sequencing NS4B-  sense TACTCCTCCATGCC 7494-751648 3′UTR region CCCCCTTGA HCV1bSR7558 sequencing NS4B-  antisenseCTCGCTCACRGTAG 7570-7548 49 3′UTR region ACCAAGACCC HCV1bSF7968sequencing NS4B- sense CTCCGTGTGGAAG 7961-7984 50 3′UTR regionGACTTGCTGGA HCV1bSR8077 sequencing NS4B- antisense TCTGGGAATACGAT8092-8070 51 3′UTR region AAGGCGAGC HCV1bSF8528 sequencing NS4B- senseAGCTCCAGGACTG 8521-8542 52 3′UTR region CACGATGCT HCV1bSR8622sequencing NS4B- antisense TACCTAGTCATAGC 8638-8615 53 3′UTR regionCTCCGTGAAG HCV1bSF9010 sequencing NS4B- sense ACACGCTGTGATAA 9010-903254 3′UTR region ATGTCTCCCCCGC HCV1bSR9094 sequencing NS4B- antisenseGATGTCTCCAGACT 9108-9087 55 3′UTR region CGCAAGGG

Other specific embodiments of the invention relate to analogous assaysfor use with samples obtained from subjects infected with HCV of othergenotypes and subtypes, in particular HCV 1a, HCV 2 and HCV 3. A personskilled in the art will know how to design appropriate cDNA synthesisand amplification primers according to the method of the invention aswell as sequencing primers by reference to prototype viral genomesequences.

Samples may consist of but are not limited to blood samples of subjectsinfected by HCV or co-infected by HIV and HCV, which samples may betaken from the subjects prior to, during or subsequent to a course oftreatment with an anti-HCV drug or drug combination. The assays of theinvention may be used for other applications. For example, HCV variantspresent in a sample from a subject may be cloned into an infectious HCVvector and transduced into mammalian cells. (Kato et as. (2007) J Virol81, 4405-4411). Infectious virus containing HCV variant sequences maythereafter be used to infect mammalian cultures, and samples obtainedafter one or several cycles of infection in the presence or absencepresence of an anti-HCV drug or drug combination may be analysed by theassays of the invention.

HCV RNA extraction from samples may be performed by various methodsincluding the use of commercially available kits, e.g., QIAamp® ViralRNA mini kit, QIAamp® Utralsens™ Virus Kit, Trizol reagens (Invitrogen)or Vivaspin concentration.

The reverse transcription and PCR reactions that are part of the assaysare performed using standard reaction mixtures and conditions such asthose described in the examples and the references cited herein.Typically, nucleic acids extracted from samples are subjected to 20-40amplification cycles in the PCR reactions of steps 4 and 2-10 cycles inthe nested PCR reaction of steps 5.

All publications and patents cited herein shall be considered asincorporated by reference in their entirety.

The invention is further elaborated by the following examples. Theexamples are provided for purpose of illustration to a person skilled inthe art, and are not intended to be limiting the scope of the inventionas described in the claims. Thus, the invention should not be construedas being limited to the examples provided, but should be construed toencompass any and all variations that become evident as a result of theteaching provided herein.

EXAMPLES Example 1 Detailed Methods used in the Assays of the Invention

Samples: Plasma samples from therapy-naive patients were obtained fromtwo different hospitals. Viral load was determined using the HCV ViralLoad COBAS AMPLICOR system from Roche Molecular Diagnostics (Basel,Switzerland).

Samples were selected based on the presence of HCV of type 1b asdetermined by InnoLipa test (Innogenetics, Zwijnaarde, Belgium). Toconfirm genotype, after cDNA synthesis and PCR amplification with oneset of outer primer pairs, sequencing was performed with one of thelatter primers. The resulting sequence was genotyped and subtyped usingthe Oxford Automated HCV Subtyping tool. (De Oliveira et al. (2005)Bioinformatics 21, 3797-3800.)

Primer selection and synthesis: Primers for reverse transcription, PCRamplification and sequencing were developed using the OLIGO software(Medprobe, Oslo, Norway). Aligned near-full length genome sequences weredownloaded from the Los Alamos HCV database(http://hcv.lanl.gov/content/sequence/HCV/ToolsOutline.html). The primersequences for HCV1b are given in Table 1. The primers were synthesisedby Applera Europe (Lennik, Belgium).

RNA extraction: RNA extraction was performed using the QIAamp Viral RNAmini kit from Qiagen (Westburg, Leusden, The Netherlands) according tothe manufacturer's protocol.

cDNA synthesis and PCR amplification: RNA was reverse-transcribed inthree separate reactions (primed by the three outer antisense primers)with Transcriptor RT from Roche (Roche Diagnostics, Mannheim, Germany).First, 2.5 μM outer antisense primer and 10 μl RNA were denatured in amicrotube for 5 min at 65° C. and snap-cooled. Next, a reversetranscription mixture was assembled in a final volume of 20 μl, themixture including the following additional components: 1× TranscriptorRT-buffer, 1 mM dNTP's, 20U of Protector RNase Inhibitor, 10U ofTranscriptor Reverse Transcriptase and MilliQ water. cDNA synthesis wasperformed at 50° C. for 90 minutes, and reactions were then cooled downto 4° C. PCR amplification was done in two steps using the Expand LongTemplate PCR System from Roche (Roche Diagnostics, Mannheim, Germany). Afirst set of PCR was performed under the following conditions: lx ExpandLong Template Buffer 1, 0.350 mM dNTP's, 0.3 μM outer sense primer, 0.3μM outer antisense primer, 3.75U Expand Long Template DNA Polymerase, 5pl template cDNA and MilliQ water in a final volume of 50 μl. Subsequentto a denaturation step at 94° C. for 2 min, 10 cycles of 10 sec at 94°C., 30 sec at 57° C., and 4 min at 68° C. were performed. Thisamplification was followed by 25 cycles of 15 sec at 94° C., 30 sec at57° C., and 4 min at 68° C., with a time increment of 20 sec/cycle and afinal elongation step of 7 min at 68° C. The reactions were then cooledto 4° C. A second set of PCR was performed under the followingconditions: 1× Expand Long Template Buffer 1, 0.350 mM dNTP's, 0.3 μMinner sense primer, 0.3 μM inner antisense primer, 3.75U Expand LongTemplate DNA Polymerase, 2 μl amplification product from the first setof PCR and MilliQ water in a final volume of 50 μl. Cycling conditionswere identical to those of the first set of PCR except that only threecycles were performed.

Gel electrophoresis: PCR products were analyzed by agarose gelelectrophoresis. 7.5 μl of PCR product were loaded on a 1.5% agarosegel, and electrophoresis was performed at 100V. Gels were stained for 10min with ethidium bromide for visualising DNA fragments.

PCR product purification: PCR products were purified using the Qiaquick

PCR purification kit from Qiagen (Westburg, Leiden, The Netherlands)according to the manufacturer's protocol. Concentration of purified PCRproducts was determined spectrophotometrically. Sequencing reactionsrequired about 2ng/100 by of DNA.

Nucleotide sequencing: Sequencing reactions were performed at FasterisSA (Geneva, Switzerland) using the BigDye Terminator v3.1 CycleSequencing kit (Applied Biosystems Inc., Foster City, USA). Contigs wereassembled and the data were analysed in Seqscape (Applied BiosystemsInc., Foster City, USA).

Example 2 Validation of Assays of the Invention: PCR Success Rate, andReproducibility and Sensitivity of an Assay of the Invention

The rate of success in obtaining visualizable and sequencable quantitiesof three overlapping PCR products representing nearly the entire viralgenome from HCV 1b-containing samples was determined using 60 samplesfrom infected subjects that were originally genotyped as HCV1b byInno-LiPA HCV I. Genotypes/subtypes were re-analyzed by sequencing andphylogenetic analysis for samples with equivocal results.

Reproducibility of the assay was assessed for each partial genome PCR bytriplicate testing of samples of confirmed genotype 1b. At least 2 ofthe 3 repeat testings were performed starting from plasma sample.Exceptionally, the 3rd repeat testing was started from the extracted RNAfor samples with insufficient plasma volumes.

Sensitivity of the assay was validated on 10 samples.

Specificity of the assay was estimated by partial sequencing of PCRproducts produced from a subset of 8 samples. All sequences were eitherHCV1b or 1a. Results of these analyses are summarized in Table 2 below.Example results of the sensitivity determinations are presented in FIG.2.

The data presented in Table 2 demonstrate that PCR success rateapproached 100% after subtype correction. The assay was HCV genotype1-specific. Good reproducibility was observed. Sensitivity was excellentfor the 5′UTR-NS2 and NS2-NS5A regions, and acceptable for the NS4B-NS5Bregion.

TABLE 2 PCR success rate, and reproducibility and sensitivity of anassay of the invention PCR PCR success success rate rate HCV InnoLipa-sequence- Sensitivity genome typed as typed as (copies/ Pairs of primersregion HCV1b HCV1b Reproducibility reaction) HCV1bOR3306/ 5′UTR-NS246/60 45/47 27/45 (3 of 3 tests) 1-10 HCVOF129 (82%) (96%) 16/45 (2 of 3tests)  2/45 (1 of 3 tests) HCV1bOR6312/ NS2-NS5A 55/60 44/47 17/44 (3of 3 tests) 10-100 HCV1bOF1977 (92%) (94%) 22/44 (2 of 3 tests)  5/44 (1of 3 tests) poly-A/ NS4B- 54/60 (45/47) 19/45 (3 of 3 tests) 100-1000HCV1bOF6074 NS5B (90%) (96%) 18/45 (2 of 3 tests)  8/45 (1 of 3 tests)

Example 3 Analysis of Samples from Treatment-Naïve, HCV-InfectedSubjects by Means of an Assay of the Invention

Aliquots of plasma samples of treatment-naïve, HCV-infected subjectswere analysed by an assay of the invention (the assay of claim 5).Nucleic acid sequences obtained after sequencing were automaticallytranslated into amino acids and compared/aligned with clustalw2.0program (at EBisite) to an HCV 1b consensus sequence, HCV coni (GenbankAccession Number AF011751). Sequences 5306 and 5415 are from twotherapy-naïve, infected subjects. Translated amino acid sequencesderived from HCV present in the two subjects are compared to the conisequence in FIGS. 3 a and 3 b. Sequence identity with the consensussequence is indicated by dash in the sequences from the infectedsubjects. The beginning of the coding sequence for each viral protein isindicated (CORE, E1, E2, P7, NS2,NS3, NS4A, NS4B, NS5A, NS5B).

The amino acid sequences derived from HCV present in the naïve, infectedsubjects reveal the presence of mutations L91 M in the core proteinassociated with resistance to IFN/Ribavirin therapy and V499A in NS5Bassociated with resistance to non-nucleoside inhibitors or benzimidazolecompounds. See Table 3 for relevant published information.

TABLE 3 Summary of some amino acid mutations found in the HCV1b genomeof subjects or HCV replicons that are associated with drug resistanceProtein region in HCV1b Mutations Drug Resistance Publication referenceCore L91M IFN/Ribavirin therapy Akuta et al, Virology, 2005 NS5B V499ANon-nucleoside Kukolj et al., JBC, inhibitor, 2005; benzimidazole Hwu etal., Antivir. compounds Res., 2008 NS5A T245A IFN/Ribavirin therapyNousbaum et al., J. in HCV 1a-infected Virology, 2000 subjects NS3C16S/C ACH806 Yang et al., Antimicrob. Agent Chem., 2008

1-8. (canceled)
 9. A pair of primers selected from the group consistingof: (a) poly-A and HCV1bO F6074; (b) HCV1bOR6312 and HCV1bOF1977; (c)HCV1bOR3306 and HCVOF129; (d) HCV1bIR9339 and HCV1bIF6126; (e)HCV1bIR6282 and HCV1bIF2523; (f) HCV1bIR2770 and HCVIF278; andcombinations thereof.
 10. The pair of primers of claim 9, wherein saidpair of primers is HCV1bOR6312 and HCV1bOF1977.
 11. The pair of primersof claim 9, wherein said pair of primers is HCV1bOR3306 and HCVOF129.12. The pair of primers of claim 9, wherein said pair of primers isHCV1bIR9339 and HCV1bIF6126.
 13. The pair of primers of claim 9, whereinsaid pair of primers is HCV1bIR6282 and HCV1bIF2523.
 14. The pair ofprimers of claim 9, wherein said pair of primers is of HCV1bIR2770 andHCVIF278.
 15. A kit for detecting mutations in an HCV genome, whereinsaid kit comprises the pair of primers of claim
 9. 16. (canceled) 17.The pair of primers of claim 9, wherein said pair of primers consists ofpoly-A and HCV1bOF6074.